PROJECT SUMMARY/ABSTRACT Osteoporosis is a common clinical condition characterized by low bone mass that increases the risk of fragility fractures in the elderly population. Since bone formation is clearly impaired in osteoporotic patients, a more complete understanding of the fundamental molecular mechanisms that regulate bone metabolism is likely to lead to the development of novel therapies. Therefore, identification of novel molecular pathways which influence bone formation is crucial to the development of clinical treatments to combat osteoporosis. Our previous work has definitively established that the anti-osteogenic transcription factor, retinoic acid receptor- related orphan receptor-beta (Ror?) plays an inhibitory role in osteoblast differentiation. Increasing expression levels of Ror? during physiological aging contributes to bone loss, as mice lacking Ror? exhibit higher bone mass through activation of the bone anabolic Wnt pathway. This leads to the notion that inhibition of Ror? may represent a novel paradigm to increase bone mass throughout the aging process. We hypothesized that microRNAs (miRs) may control Ror? levels, and found that a specific miR, miR-219a-5p (miR-219a), the top predicted Ror?-regulatory miR, exhibits an inverse expression pattern to Ror?, suggesting a potential Ror?/miR-219a regulatory axis in bone. We demonstrate that miR-219a directly regulates Ror? levels in osteoblasts, that delivery of miR-219a mimics enhances osteoblast differentiation, and that miR-219a antagonists suppress differentiation. We further show that miR-219a increases the activity of the bone anabolic Wnt pathway. Therefore, we propose that miR-219a is bone anabolic and may be used to preserve bone mass with age, possibly through increasing Wnt pathway activity. Using a novel mouse model where we can activate miR-219a expression in a tissue-specific manner, in Aim 1 we will determine the effects of miR-219a in Ror?- expressing cells on adult bone mass or following a fracture, with the hypothesis that downregulation of Ror? through miR-219a will increase bone mass and accelerate fracture healing. In Aim 2, we will explore the more general effects of miR-219a in bone homeostasis by activating miR-219a in various bone cell lineages, with the hypothesis that downregulation of Ror?, and/or other miR-219a targets in these lineages, will increase bone mass with age. Finally, in Aim 3 we will perform in vitro experiments to explore the molecular mechanism of how miR-219a regulates the Wnt pathway and determine whether direct targeting of Ror? by miR-219a affects Wnt activity and bone cell function. Completion of these pivotal studies will not only provide a more complete understanding of miR-219a function in bone, but if positive will also provide a strong justification for pursuing miR-219a mimics as a novel therapeutic strategy to stimulate bone formation in various clinical conditions.